1. Field of the Invention
The present invention relates to methods for identifying chemicals that act as agonists or antagonists for proteins participating in signal transduction pathways that utilize heterotrimeric guanine nucleotide-binding proteins (G-proteins) and/or second messengers, e.g., cyclic adenosine monophosphate (cAMP) and to a method for identifying nucleic acid clones coding for G-protein coupled cell surface receptors (GPC receptors) that act via signal transduction pathways that utilize G-proteins and/or second messengers, e.g., cAMP.
2. Background Information
All publications referred to herein are incorporated by reference.
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz (1991) Nature, 351: 353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors such as those for adrenergic agents and dopamine (Kobilka B. K., Dixon, R. A., Frielle, T., et al. (1987) Proc Natl Acad Sci U S A 84: 46-50; Kobilka, B. K., Matsui, H., Kobilka, T. S., et al. (1987) Science 238: 650-656; Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., et al. (1988) Nature 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I.; Strathmann, M. P.; Gautam, N., (1991) Science 252: 802-8).
Bioassays for chemicals that activate a few GPC receptors that are endogenous in pigment cells are described in Negishi et al. (1988), General and Comparative Endocrinology, 70: 127-132; Messenger and Warner (1977) Br. J. Pharmacology 61:607-614; Mori and Lerner, (1960) Endocrinology 67: 443-450; Moller and Lerner (1966) Acta Endocrinologica, 51: 149-160; Carter and Shuster (1978) J. Inv. Dermatology, 71:229-232; Lerner et al. (1988), P.N.A.S. USA 85: 261-264; and C. H. Elwing et al. (1990), Biosensors & Bioelectronics, 5: 449-459.
In all of the methods described in the publications listed above, there are the following six major differences between them and the applicants' methods:
(1) The applicants' methods are based on pigment cells that can be grown in continuous long term culture whereas none of the bioassays described in the above publications makes use of pigment cells that continue to divide in culture. The advantage of the applicants' method is that it allows for the straightforward generation of an unlimited number of cells to be used for assays. Without this ability, large scale drug screens are not possible.
(2) Only the applicants' methods allow for a continuous source of pigment cells generated from existing ones without the need to collect fresh cells from animals.
(3) Only the applicants' methods utilize pigment cells that can be grown to high density in tissue culture vessels. This is important for the ability to screen large numbers of drugs and it is important for the ability to being able to obtain reliable results using standard microtiter plate readers.
(4) The applicants' methods can be used to screen for drugs which affect the endogenous serotonin receptor on pigment cells which causes pigment dispersion. In contrast thereto, in the publications listed above, serotonin is stated to cause pigment aggregation, e.g., Messenger and Warner (1977) Br. J. Pharmacology 61:607-614.
(5) The applicants' methods utilize recombinant DNA technology so that the pigment cells can serve as the basis of drug assays for receptors and other proteins that are not naturally expressed by pigment cells. The methods described in the above publications, however, are limited to receptors that are endogenous to pigment cells.
(6) In contrast to the above-described publications, only the applicants' methods can be used to clone GPC receptors because the applicants' methods make use of continuous cultures of pigment cells and recombinant DNA technology.
Currently there is a major limitation in finding new and better drugs for GPC receptors, namely, no initial screen exists for testing the abilities of chemicals to affect GPC receptors that is simple, rapid and general. For example, consider assays for evaluating GPC receptors that work via Gs or Gi to raise or lower intercellular cAMP. Radioimmunoassay (RIA) for cAMP accumulation is both expensive, slow and a single technician would be hardpressed to screen more than 20 chemicals in triplicate in a single day (Steiner et al. (1972) J. Biol. Chem., 247: 1106-1113). Meanwhile, the current adenylate cyclase activation assay is faster than the RIA, and a single individual can process up to 150 samples in a day (Salomon et al. (1974) Analytical Biochemistry 58: 541-548). However, the current adenylate cyclase activation assay requires several steps and is therefore cumbersome. Also, both procedures involve substantial use of radioactive materials, for example, either .sup.32 P or .sup.125 I.
Definitions
A "chemical" is defined to include any drug, compound or molecule.
A "G-protein coupled cell surface receptor" (GPC receptor) is defined to be any cell surface transmembrane protein, that when activated by a chemical, in turn activates a heterotrimeric guanine nucleotide-binding protein (G-protein).
A "protein participating in a signal transduction pathway that involves a G-protein and/or a second messenger (PPG protein)" is defined as any protein involved in the pathway including GPC receptors, G-proteins, effector proteins and actuator proteins.
An "effector protein" is defined as any protein which is activated or inactivated by an .alpha. subunit of a G-protein. Some examples of effector proteins include adenyl cyclase, phospholipase C and phospholipase A2. Phosphodiesterase is also considered an effector protein.
A "second messenger" is defined as an intermediate compound whose concentration, either intercellularly or within the surrounding cell membrane, is raised or lowered as a consequence of the activity of an effector protein. Some examples of second messengers include cyclic adenosine monophosphate (cAMP), phosphotidyl inositols (PI), such as inositol triphosphate (IP3), diacylglycerol (DAG), calcium (Ca++) and arachidonic acid derivatives.
An "actuator protein" is defined as a protein whose state of activation is modified as a result of binding a second messenger. Some examples of effector proteins include protein kinase A and protein kinase C.
A schematic example that provides a summary of the above definitions by example of one pathway that utilizes G-proteins and the second messenger cAMP is given in FIG. 1.
"Pigment cells" mean any pigment-containing cells that meet the following conditions: (1) They are derived from any animal whose pigment cells are capable of aggregating or dispersing their pigment in response to a specific stimulus, e.g., contact with melanocyte stimulating hormone, melatonin, light, etc, (2) They can be indefinitely propagated in vitro so that unlimited quantities of cells can be obtained. (3) Pigment cells ("test cells") for use in the present invention include the following non-limiting examples of chromatophores: melanophores or melanocytes, xanthophores, erythrophores, leukophores and iridophores. The pigment cells are taken from animals lower on the evolutionary tree than humans and birds. Non-limiting examples of "lower animals" from which pigment cells can be taken for utilization in the present invention include the following: Reptilia, e.g., Anolis sp; Amphibia, e.g., Xenopus laevis; Pisces, e.g., Zacco temmincki; Crustacia, e.g., Uca pugilator; Echinodermata, e.g., Diadema antillarum and Cinidaria, e.g., Nanomsa cara. Particularly preferred pigment cells for use in the present invention are cultured melanophores from the from Xenopus laevis (Pigment Cell 1985), ed. Bagnara et al., University of Tokyo Press, pages 219-227) and Lerner et al. (1988) P.N.A.S. USA, 85: 261-264.